Promoter, promoter control elements, and combinations, and uses thereof

ABSTRACT

The present invention is directed to promoter sequences and promoter control elements, polynucleotide constructs comprising the promoters and control elements, and methods of identifying the promoters, control elements, or fragments thereof. The invention further relates to the use of the present promoters or promoter control elements to modulate transcript levels.

This application is a Divisional of co-pending application Ser. No.11/602,163 filed on Nov. 20, 2006, and for which priority is claimedunder 35 U.S.C. §120; and this application claims priority of U.S.application Ser. No. 11/172, 703, filed on Jun. 30, 2005, under 35U.S.C. §119; which claims priority to Provisional Application No(s).60/583,691 and 60/583,609 both filed on Jun. 30, 2004, the entirecontents of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to promoters and promoter control elementsthat are useful for modulating transcription of a desiredpolynucleotide. In order to modulate in vivo and in vitro transcriptionof a polynucleotide such promoters and promoter control elements can beincluded in polynucleotide constructs, expression cassettes, vectors orinserted into the chromosome or exist in the plant cell as an exogenouselement. Host cells with polynucleotides comprising the promoters andpromoter control elements of the present invention which have desiredtraits or characteristics resulting therefrom are also a part of theinvention. This includes plant cells and plants regenerated therefrom.

BACKGROUND OF THE INVENTION

This invention relates to the field of biotechnology and in particularto specific promoter sequences and promoter control element sequenceswhich are useful for the transcription of polynucleotides in a host cellor transformed host organism.

One of the primary goals of biotechnology is to obtain organisms such asplants, mammals, yeast and prokaryotes that have particular desiredcharacteristics or traits. Examples of these characteristics or traitsabound and in plants may include, for example, virus resistance, insectresistance, herbicide resistance, enhanced stability, enhanced biomass,enhanced yield or additional nutritional value.

Recent advances in genetic engineering have enabled researchers in thefield to incorporate polynucleotide sequences into host cells to obtainthe desired qualities in the organism of choice. This technology permitsone or more polynucleotides from a source different than the organism ofchoice to be transcribed by the organism of choice. If desired, thetranscription and/or translation of these new polynucleotides can bemodulated in the organism to exhibit a desired characteristic or trait.Alternatively, new patterns of transcription and/or translation ofpolynucleotides endogenous to the organism can be produced. Bothapproaches can be used at the same time.

SUMMARY OF THE INVENTION

The present invention is directed to isolated polynucleotide sequencesthat comprise promoters and promoter control elements from plants,especially Arabidopsis thaliana, Glycine max, Oryza sativa and Zea mays,and other promoters and promoter control elements that function inplants.

It is an object of the present invention to provide isolatedpolynucleotides that are promoter sequences. These promoter sequencescomprise, for example,

-   (1) a polynucleotide having a nucleotide sequence as set forth in    the Sequence Listing or a fragment thereof,-   (2) a polynucleotide having a nucleotide sequence having at least    80% sequence identity to a sequence as set in the Sequence Listing    or a fragment thereof, and-   (3) a polynucleotide having a nucleotide sequence which hybridizes    to a sequence as set forth in in the Sequence Listing under a    condition establishing a Tm-20° C.

It is another object of the present invention to provide isolatedpolynucleotides that are promoter control element sequences. Thesepromoter control element sequences comprise, for example,

-   (1) a polynucleotide having a nucleotide sequence as set forth in    the Sequence Listing or a fragment thereof,-   (2) a polynucleotide having a nucleotide sequence having at least    80% sequence identity to a sequence as set forth in the Sequence    Listing or a fragment thereof, and-   (3) a polynucleotide having a nucleotide sequence which hybridizes    to a sequence as set forth in the Sequence Listing under a condition    establishing a Tm-20° C.

Promoter or promoter control element sequences of the present inventionare capable of modulating preferential transcription.

In another embodiment, the present promoter control elements are capableof serving as or fulfilling the function of, for example, a corepromoter, a TATA box, a polymerase binding site, an initiator site, atranscription binding site, an enhancer, an inverted repeat, a locuscontrol region, or a scaffold/matrix attachment region.

It is yet another object of the present invention to provide apolynucleotide that includes at least a first and a second promotercontrol element. The first promoter control element is a promotercontrol element sequence as discussed above and the second promotercontrol element is heterologous to the first control element. Moreover,the first and second control elements are operably linked. Suchpromoters may modulate transcript levels preferentially in a tissue orunder particular conditions.

In another embodiment, the present isolated polynucleotide comprises apromoter or a promoter control element as described above, wherein thepromoter or promoter control element is operably linked to apolynucleotide to be transcribed.

In another embodiment of the present vector, the promoter and promotercontrol elements of the instant invention are operably linked to aheterologous polynucleotide that is a regulatory sequence.

It is another object of the present invention to provide a host cellcomprising an isolated polynucleotide or vector as described above or afragment thereof. Host cells include, for instance, bacterial, yeast,insect cells, mammalian cells and plant cells. The host cell cancomprise a promoter or promoter control element exogenous to the genome.Such a promoter can modulate transcription in cis- and intrans-orientation to the polynucletide.

In yet another embodiment, the present host cell is a plant cell capableof regenerating into a plant.

It is yet another embodiment of the present invention to provide a plantcomprising an isolated polynucleotide or vector described above.

It is another object of the present invention to provide a method ofmodulating transcription in a sample that contains either a cell-freesystem of transcription or a host cell. This method comprises providinga polynucleotide or vector according to the present invention asdescribed above and contacting the sample of the polynucleotide orvector with conditions that permit transcription.

In another embodiment of the present method, the polynucleotide orvector preferentially modulates

-   (a) constitutive transcription,-   (b) stress induced transcription,-   (c) light or shade induced transcription,-   (d) dark induced transcription,-   (e) leaf transcription,-   (f) root transcription,-   (g) stem or shoot transcription,-   (h) silique or seed transcription,-   (i) callus transcription,-   (j) flower transcription,-   (k) immature bud and inflorescence-specific transcription, or-   (l) senescence induced transcription-   (m) germination transcription.    Other and further objects of the present invention will be made    clear or become apparent from the following description.

BRIEF DESCRIPTION OF THE TABLES AND FIGURES Table 1

Table 1 consists of the Expression Reports for each promoter of theinvention and provides the nucleotide sequence for each promoter as wellas details for GFP expression driven by each of the nucleic acidpromoter sequences as observed in transgenic plants. The results arepresented as summaries of the spatial expression, which providesinformation as to gross and/or specific expression in various plantorgans and tissues. The observed expression pattern is also presented,which gives details of expression during different generations ordifferent developmental stages within a generation. Additionalinformation is provided regarding the associated gene, the GenBankreference, the source organism of the promoter and the vector and markergenes used for the construct. The following symbols are usedconsistently throughout the Table:

-   T1: First generation transformant-   T2: Second generation transformant-   T3: Third generation transformant-   (L): low expression level-   (M): medium expression level-   (H): high expression level

Each row of the table begins with heading of the data to be found in thesection. The following provides a description of the data to be found ineach section:

Heading in Table 1 Description Promoter Expression Report Identifies theparticular promoter report Promoter tested in Identifies the organismused for analysis Spatial expression summary: Identifies the organs andtissues where expression was observed and estimates the strength ofexpression Observed expression pattern: Presents expression patternobserved for various generations of plants and developmental stagesExpected expression pattern: Identifies the pattern expected from otherexperiments Selection Criteria: Provides details on cloning thepolynucleotide Gene: Provides information concerning the gene modulatedby the promoter GenBank: This field gives the Locus Number of the geneas well as the accession number. Source Promoter Organism: Identifiesthe organism from which the promoter was cloned. Vector: Identifies thevector into which the promoter was cloned. Marker Type: Identifies thetype of marker linked to the promoter. The marker is used to determinepatterns of gene expression in plant tissue. Generation screened: T1Mature T2 Identifies the plant generation(s) used in the Seedling T2Mature T3 Seedling screening process. T1 plants are those plantssubjected to the transformation event while the T2 generation plants arefrom the seeds collected from the T1 plants and T3 plants are from theseeds of T2 plants. Plant Expression Identifies the generation anddevelopmental stage of the plants analyzed Events Screened EventsExpressing Provides the number of independent transformation eventsanalyzed and the number which expressed the marker gene GFP ExpressionDetected This section lists the various organs analyzed and, whereexpression was observed, indicates the strength of the expression X inthe . . . This field summarizes the expression pattern from digitalimages of the cells Promoter Utility: Identifies a specific function orfunctions that can be modulated using the promoter cDNA. Trait-SubtraitArea: Provides information as to what agronomic traits could be alteredConstruct: Provides the Ceres identifier number for the constructPromoter Candidate I.D.: Provides the Ceres identifier number for thepromoter isolated cDNA ID: Provides the Ceres identifier numberassociated with the cDNA that corresponds to the endogenous cDNAsequence of the promoter. T1 lines expressing (T2 seed): Provides theidentifier numbers for the events analyzed Sequence Provides thenucleotide sequence for the promoter described in the report

Table 2

Table 2 provides a partial summary of the expression for some of theconstructs of the invention.

FIG. 1 is a schematic representation of the vector pNewBin4-HAP1-GFP.The definitions of the abbreviations used in the vector map are asfollows:

-   Ori—the origin of replication used by an E. coli host-   RB—sequence for the right border of the T-DNA from pMOG800-   BstXI—restriction enzyme cleavage site used for cloning-   HAP1VP16—coding sequence for a fusion protein of the HAP1 and VP16    activation domains-   NOS—terminator region from the nopaline synthase gene-   HAP1UAS—the upstream activating sequence for HAP1-   5ERGFP—the green fluorescent protein gene that has been optimized    for localization to the endoplasmic reticulum-   OCS2—the terminator sequence from the octopine synthase 2 gene-   OCS—the terminator sequence from the octopine synthase gene-   p28716 (a.k.a 28716 short)—promoter used to drive expression of the    PAT (BAR) gene-   PAT (BAR)—a marker gene conferring herbicide resistance-   LB—sequence for the left border of the T-DNA from pMOG800-   Spec—a marker gene conferring spectinomycin resistance-   TrfA—transcription repression factor gene-   RK2-OriV—origin of replication for Agrobacterium

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Chimeric: The term “chimeric” is used to describe polynucleotides orgenes, as defined supra, or constructs wherein at least two of theelements of the polynucleotide or gene or construct are heterologous toeach other, such as the promoter and the polynucleotide to betranscribed and/or other regulatory sequences and/or filler sequencesand/or complements thereof.

Constitutive Promoter: Promoters referred to herein as “constitutivepromoters” actively promote transcription under most, but notnecessarily all, environmental conditions and is essentially all cellsin the vegetative stage and/or flowers and essentially all states ofcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcript initiation region and the1′ or 2′ promoter derived from T-DNA of Agrobacterium tumefaciens aswell as other transcription initiation regions from various plant genesknown to those of skill in the art, such as the maize ubiquitin-1promoter.

Core Promoter: This is the minimal stretch of contiguous DNA sequencethat is sufficient to direct accurate initiation of transcription by theRNA polymerase II machinery (for review see: Struhl, 1987, Cell 49:295-297; Smale, 1994, In Transcription: Mechanisms and Regulation (edsR. C. Conaway and J. W. Conaway), pp 63-81/Raven Press, Ltd., New York;Smale, 1997, Biochim. Biophys. Acta 1351: 73-88; Smale et al., 1998,Cold Spring Harb. Symp. Quant. Biol. 58: 21-31; Smale, 2001, Genes &Dev. 15: 2503-2508; Weis and Reinberg, 1992, FASEB J. 6: 3300-3309;Burke et al., 1998, Cold Spring Harb. Symp. Quant. Biol 63: 75-82).There are several sequence motifs, including the TATA box, initiator(Inr), TFIIB recognition element (BRE) and downstream core promoterelement (DPE), that are commonly found in core promoters. Not all ofthese elements, however, occur in all promoters. That is, there are nouniversal core promoter elements (Butler and Kadonaga, 2002, Genes &Dev. 16: 2583-2592).

Domain: Domains are fingerprints or signatures that can be used tocharacterize protein families and/or parts of proteins. Suchfingerprints or signatures can comprise conserved (1) primary sequence,(2) secondary structure, and/or (3) three-dimensional conformation. Asimilar analysis can be applied to polynucleotides. Generally, eachdomain has been associated with either a conserved primary sequence or asequence motif. Generally these conserved primary sequence motifs havebeen correlated with specific in vitro and/or in vivo activities. Adomain can be any length, including the entirety of the polynucleotideto be transcribed. Examples of amino acid domains include, withoutlimitation, AP2, helicase, homeobox, zinc finger, etc. Examples ofnucleotide domains include, without limitation, TATA box, CAAT box, etc.

Endogenous: The term “endogenous” within the context of the currentinvention refers to any polynucleotide, polypeptide or protein sequencewhich is a natural part of a cell or organism regenerated from saidcell. In the context of promoter, the term “endogenous coding region” or“endogenous cDNA” refers to the coding region that is naturally operablylinked to the promoter.

Enhancer/Suppressor: An “enhancer” is a DNA regulatory element that canincrease the steady state level of a transcript, usually by increasingthe rate of transcription initiation. Enhancers usually exert theireffect regardless of the distance, upstream or downstream location, ororientation of the enhancer relative to the start site of transcription.In contrast, a “suppressor” is a corresponding DNA regulatory elementthat decreases the steady state level of a transcript, again usually byaffecting the rate of transcription initiation. The essential activityof enhancer and suppressor elements is to bind a protein factor(s). Suchbinding can be assayed, for example, by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in an in vitro transcription extract.

Exogenous: As referred to within, “exogenous” is any polynucleotide,polypeptide or protein sequence, whether chimeric or not, that isintroduced, into the genome of a host cell or organism regenerated fromsaid host cell by any means other than by a sexual cross. Examples ofmeans by which this can be accomplished are described below and includeAgrobacterium-mediated transformation (of dicots—e.g. Salomon et al.EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO J. 2:987 (1983); ofmonocots, representative papers are those by Escudero et al., Plant J.10:355 (1996), Ishida et al., Nature Biotechnology 14:745 (1996), May etal., Bio/Technology 13:486 (1995)), biolistic methods (Armaleo et al.,Current Genetics 17:97 1990)), electroporation, in planta techniques andthe like. Such a plant containing the exogenous nucleic acid is referredto here as a T₀ for the primary transgenic plant and T₁ for the firstgeneration transformant. The term “exogenous” as used herein is alsointended to encompass inserting a naturally found element into anon-naturally found location.

Gene: The term “gene,” as used in the context of the current invention,encompasses all regulatory and coding sequence contiguously associatedwith a single hereditary unit with a genetic function (see SCHEMATIC 1).Genes can include non-coding sequences that modulate the geneticfunction that include, but are not limited to, those that specifypolyadenylation, to transcriptional regulation, DNA conformation,chromatin conformation, extent and position of base methylation andbinding sites of proteins that control all of these. Genes encodingproteins are comprised of “exons” (coding sequences), which may beinterrupted by “introns” (non-coding sequences). In some instancescomplexes of a plurality of protein or nucleic acids or other molecules,or of any two of the above, may be required for a gene's function. Onthe other hand a gene's genetic function may require only RNA expressionor protein production, or may only require binding of proteins and/ornucleic acids without associated expression. In certain cases, genesadjacent to one another may share sequence in such a way that one genewill overlap the other. A gene can be found within the genome of anorganism, in an artificial chromosome, in a plasmid, in any other sortof vector, or as a separate isolated entity.

Heterologous Sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequenceoriginates from, are considered heterologous to said coding sequence.Elements operatively linked in nature and contiguous to each other arenot heterologous to each other.

Homologous: In the current invention, a “homologous” gene orpolynucleotide or polypeptide refers to a gene or polynucleotide orpolypeptide that shares sequence similarity with the gene orpolynucleotide or polypeptide of interest. This similarity may be inonly a fragment of the sequence and often represents a functional domainsuch as, examples including without limitation a DNA binding domain or adomain with tyrosine kinase activity. The functional activities ofhomologous polynucleotide are not necessarily the same.

Inducible Promoter: An “inducible promoter” in the context of thecurrent invention refers to a promoter the activity of which isinfluenced by certain conditions such as light, temperature, chemicalconcentration, protein concentration, conditions in an organism, cell,or organelle, etc. A typical example of an inducible promoter, which canbe utilized with the polynucleotides of the present invention, isPARSK1, the promoter from an Arabidopsis gene encoding aserine-threonine kinase enzyme which is induced by dehydration,abscissic acid and sodium chloride (Wang and Goodman, Plant J. 8:37(1995)). Examples of environmental conditions that may affecttranscription by inducible promoters include anaerobic conditions,elevated temperature, the presence or absence of a nutrient or otherchemical compound and/or the presence of light.

Modulate Transcription Level: As used herein, the phrase “modulatetranscription” describes the biological activity of a promoter sequenceor promoter control element. Such modulation includes, withoutlimitation, up- and down-regulation of initiation of transcription, rateof transcription and/or transcription levels.

Mutant: In the current invention “mutant” refers to a heritable changein a mutation sequence at a specific location. Mutant genes of thecurrent invention may or may not have an associated identifiablephenotype.

Operable Linkage: An “operable linkage” is a linkage in which a promotersequence or promoter control element is connected to a polynucleotidesequence(s) in such a way as to place transcription of thepolynucleotide sequence under the influence or control of the promoteror promoter control element. Two DNA sequences (such as a polynucleotideto be transcribed and a promoter sequence linked to the 5′ end of thepolynucleotide to be transcribed) are said to be operably linked ifinduction of promoter function results in the transcription of mRNAencoded by the polynucleotide and if the nature of the linkage betweenthe two DNA sequences does not (1) result in the introduction of aframe-shift mutation, (2) interfere with the ability of the promotersequence to direct the expression of the protein, antisense RNA orribozyme or (3) interfere with the ability of the DNA template to betranscribed. Thus, a promoter sequence would be operably linked to apolynucleotide sequence if the promoter was capable of effectingtranscription of that polynucleotide sequence.

Optional Promoter Fragments: The phrase “optional promoter fragments” isused to refer to any sub-sequence of the promoter that is not requiredfor driving transcription of an operationally linked coding region.These fragments comprise the 5′ UTR and any exon(s) of the endogenouscoding region. The optional promoter fragments may also comprise anyexon(s) and the 3′ or 5′ UTR of the gene residing upstream of thepromoter (that is, 5′ to the promoter). Optional promoter fragments alsoinclude any intervening sequences that are introns or sequence thatoccurs between exons or an exon and the UTR.

Orthologous: “Orthologous” is a term used herein to describe arelationship between two or more polynucleotides or proteins. Twopolynucleotides or proteins are “orthologous” to one another if theyserve a similar function in different organisms. In general, orthologouspolynucleotides or proteins will have similar catalytic functions (whenthey encode enzymes) or will serve similar structural functions (whenthey encode proteins or RNA that form part of the ultrastructure of acell). Generally it is believed that orthologous structures share acommon evolutionary origin.

Percentage of Sequence Identity: “Percentage of sequence identity,” asused herein, is determined by comparing two optimally aligned sequencesover a comparison window, where the fragment of the polynucleotide oramino acid sequence in the comparison window may comprise additions ordeletions (e.g., gaps or overhangs) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981),by the homology alignment algorithm of Needleman and Wunsch J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson andLipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis.), or by inspection. Giventhat two sequences have been identified for comparison, GAP and BESTFITare preferably employed to determine their optimal alignment. Typically,the default values of 5.00 for gap weight and 0.30 for gap weight lengthare used. The term “substantial sequence identity” betweenpolynucleotide or polypeptide sequences refers to polynucleotide orpolypeptide comprising a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90% and mostpreferably at least 95%, even more preferably, at least 96%, 97%, 98% or99% sequence identity compared to a reference sequence using theprograms.

Query nucleic acid sequences were searched against subject nucleic acidsequences residing in public or proprietary databases. Such searcheswere done using the Washington University Basic Local Alignment SearchTool Version 1.83 (WU-Blast2) program. The WU-Blast2 program isavailable on the internet from Washington University. A WU-Blast2service for Arabidopsis can also be found on the internet. Typically thefollowing parameters of WU-Blast2 were used: Filter options were set to“default,” Output format was set to “gapped alignments,” the ComparisonMatrix was set to “BLOSUM62,” Cutoff Score (S value) was set to“default,” the Expect (E threshold) was set to “default,” the Number ofbest alignments to show was set to “100,” and the “Sort output” optionwas set to sort the output by “pvalue.”

Plant Promoter: A “plant promoter” is a promoter capable of initiatingtranscription in plant cells and can modulate transcription of apolynucleotide. Such promoters need not be of plant origin. For example,promoters derived from plant viruses, such as the CaMV35S promoter orfrom Agrobacterium tumefaciens such as the T-DNA promoters, can be plantpromoters. A typical example of a plant promoter of plant origin is themaize ubiquitin-1 (ubi-1) promoter known to those of skill in the art.

Plant Tissue: The term “plant tissue” includes differentiated andundifferentiated tissues or plants, including but not limited to roots,stems, shoots, cotyledons, epicotyl, hypocotyl, leaves, pollen, seeds,tumor tissue and various forms of cells in culture such as single cells,protoplast, embryos, and callus tissue. The plant tissue may be inplants or in organ, tissue or cell culture.

Preferential Transcription: “Preferential transcription” is defined astranscription that occurs in a particular pattern of cell types ordevelopmental times or in response to specific stimuli or combinationthereof. Non-limitive examples of preferential transcription include:high transcript levels of a desired sequence in root tissues; detectabletranscript levels of a desired sequence in certain cell types duringembryogenesis; and low transcript levels of a desired sequence underdrought conditions. Such preferential transcription can be determined bymeasuring initiation, rate, and/or levels of transcription.

Promoter: A “promoter” is a DNA sequence that directs the transcriptionof a polynucleotide. Typically a promoter is located in the 5′ region ofa polynucleotide to be transcribed, immediately upstream to thetranscriptional start site of such polynucleotide. More typically,promoters are defined as the region upstream of the first exon; moretypically, as a region upstream of the first of multiple transcriptionstart sites; more typically, as the region downstream of the precedinggene and upstream of the first of multiple transcription start sites;more typically, the region downstream of the polyA signal and upstreamof the first of multiple transcription start sites; even more typically,about 3,000 nucleotides upstream of the ATG of the first exon; even moretypically, 2,000 nucleotides upstream of the first of multipletranscription start sites. The promoters of the invention comprise atleast a core promoter as defined above. Frequently promoters are capableof directing transcription of genes located on each of the complementaryDNA strands that are 3′ to the promoter. Stated differently, manypromoters exhibit bidirectionality and can direct transcription of adownstream gene when present in either orientation (i.e. 5′ to 3′ or 3′to 5′ relative to the coding region of the gene). Additionally, thepromoter may also include at least one control element such as anupstream element. Such elements include UARs and optionally, other DNAsequences that affect transcription of a polynucleotide such as asynthetic upstream element.

Promoter Control Element: The term “promoter control element” as usedherein describes elements that influence the activity of the promoter.Promoter control elements include transcriptional regulatory sequencedeterminants such as, but not limited to, enhancers, scaffold/matrixattachment regions, TATA boxes, transcription start locus controlregions, UARs, URRs, other transcription factor binding sites andinverted repeats.

Public Sequence: The term “public sequence,” as used in the context ofthe instant application, refers to any sequence that has been depositedin a publicly accessible database prior to the filing date of thepresent application. This term encompasses both amino acid andnucleotide sequences. Such sequences are publicly accessible, forexample, on the BLAST databases on the NCBI FTP web site (accessible viathe worldwide web). The database at the NCBI FTP site uses “gi” numbersassigned by NCBI as a unique identifier for each sequence in thedatabase, thereby providing a non-redundant database for sequence fromvarious databases, including GenBank, EMBL, DBBJ, (DNA Database ofJapan) and PDB (Brookhaven Protein Data Bank).

Regulatory Sequence: The term “regulatory sequence,” as used in thecurrent invention, refers to any nucleotide sequence that influencestranscription or translation initiation and rate, or stability and/ormobility of a transcript or polypeptide product. Regulatory sequencesinclude, but are not limited to, promoters, promoter control elements,protein binding sequences, 5′ and 3′ UTRs, transcriptional start sites,termination sequences, polyadenylation sequences, introns, certainsequences within amino acid coding sequences such as secretory signals,protease cleavage sites, etc.

Related Sequences: “Related sequences” refer to either a polypeptide ora nucleotide sequence that exhibits some degree of sequence similaritywith a reference sequence.

Specific Promoters: In the context of the current invention, “specificpromoters” refers to a subset of promoters that have a high preferencefor modulating transcript levels in a specific tissue or organ or celland/or at a specific time during development of an organism. By “highpreference” is meant at least 3-fold, preferably 5-fold, more preferablyat least 10-fold still more preferably at least 20-fold, 50-fold or100-fold increase in transcript levels under the specific condition overthe transcription under any other reference condition considered.Typical examples of temporal and/or tissue or organ specific promotersof plant origin that can be used with the polynucleotides of the presentinvention, are: PTA29, a promoter which is capable of driving genetranscription specifically in tapetum and only during anther development(Koltonow et al., Plant Cell 2:1201 (1990); RCc2 and RCc3, promotersthat direct root-specific gene transcription in rice (Xu et al., PlantMol. Biol. 27:237 (1995); and TobRB27, a root-specific promoter fromtobacco (Yamamoto et al., Plant Cell 3:371 (1991)). Examples oftissue-specific promoters under developmental control include promotersthat initiate transcription only in certain tissues or organs, such asroot, ovule, fruit, seeds, or flowers. Other specific promoters includethose from genes encoding seed storage proteins or the lipid bodymembrane protein, oleosin. A few root-specific promoters are notedabove. See also “Preferential transcription”.

Stringency: “Stringency” as used herein is a function of probe length,probe composition (G+C content) and salt concentration, organic solventconcentration and temperature of hybridization or wash conditions.Stringency is typically compared by the parameter T_(m), which is thetemperature at which 50% of the complementary molecules in thehybridization are hybridized, in terms of a temperature differentialfrom T_(m). High stringency conditions are those providing a conditionof T_(m)−5° C. to T_(m)−10° C. Medium or moderate stringency conditionsare those providing T_(m)−20° C. to T_(m)−29° C. Low stringencyconditions are those providing a condition of T_(m)−40° C. to T_(m)−48°C. The relationship of hybridization conditions to T_(m) (in ° C.) isexpressed in the mathematical equation

T _(m)=81.5−16.6(log₁₀ [Na⁺])+0.41(% G+C)−(600/N)  (1)

where N is the length of the probe. This equation works well for probes14 to 70 nucleotides in length that are identical to the targetsequence. The equation below for T_(m) of DNA-DNA hybrids is useful forprobes in the range of 50 to greater than 500 nucleotides, and forconditions that include an organic solvent (formamide).

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41 (% G+C)−500/L 0.63 (%formamide)  (2)

where L is the length of the probe in the hybrid (P. Tijessen,“Hybridization with Nucleic Acid Probes” in Laboratory Techniques inBiochemistry and Molecular Biology, P.C. vand der Vliet, ed., c. 1993 byElsevier, Amsterdam.) The T_(m) of equation (2) is affected by thenature of the hybrid; for DNA-RNA hybrids T_(m) is 10-15° C. higher thancalculated, for RNA-RNA hybrids T_(m) is 20-25° C. higher. Because theT_(m) decreases about 1° C. for each 1% decrease in homology when a longprobe is used (Bonner et al. (1973) J. Mol. Biol: 81:123), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation (2) is derived assuming equilibrium and therefore,hybridizations according to the present invention are most preferablyperformed under conditions of probe excess and for sufficient time toachieve equilibrium. The time required to reach equilibrium can beshortened by inclusion of a hybridization accelerator such as dextransulfate or another high volume polymer in the hybridization buffer.

Stringency can be controlled during the hybridization reaction or afterhybridization has occurred by altering the salt and temperatureconditions of the wash solutions used. The formulas shown above areequally valid when used to compute the stringency of a wash solution.Preferred wash solution stringencies lie within the ranges stated above;high stringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

Substantially Free of: A composition containing A is “substantially freeof” B when at least 85% by weight of the total A+B in the composition isA. Preferably, A comprises at least about 90% by weight of the total ofA+B in the composition, more preferably at least about 95% or even 99%by weight. For example, a plant gene can be substantially free of otherplant genes. Other examples include, but are not limited to, ligandssubstantially free of receptors (and vice versa), a growth factorsubstantially free of other growth factors and a transcription bindingfactor substantially free of nucleic acids.

Suppressor: See “Enhancer/Suppressor”

TATA to Start: “TATA to start” shall mean the distance, in number ofnucleotides, between the primary TATA motif and the start oftranscription.

Transgenic Plant: A “transgenic plant” is a plant having one or moreplant cells that contain at least one exogenous polynucleotideintroduced by recombinant nucleic acid methods.

Translational Start Site: In the context of the present invention, a“translational start site” is usually an ATG or AUG in a transcript,often the first ATG or AUG. A single protein encoding transcript,however, may have multiple translational start sites.

Transcription Start Site: “Transcription start site” is used in thecurrent invention to describe the point at which transcription isinitiated. This point is typically located about 25 nucleotidesdownstream from a TFIID binding site, such as a TATA box. Transcriptioncan initiate at one or more sites within the gene and a singlepolynucleotide to be transcribed may have multiple transcriptional startsites, some of which may be specific for transcription in a particularcell-type or tissue or organ. “+1” is stated relative to thetranscription start site and indicates the first nucleotide in atranscript.

Upstream Activating Region (UAR): An “Upstream Activating Region” or“UAR” is a position or orientation dependent nucleic acid element thatprimarily directs tissue, organ, cell type, or environmental regulationof transcript level, usually by affecting the rate of transcriptioninitiation. Corresponding DNA elements that have a transcriptioninhibitory effect are called herein “Upstream Repressor Regions” or“URR”s. The essential activity of these elements is to bind a proteinfactor. Such binding can be assayed by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in vitro transcription extract.

Untranslated Region (UTR): A “UTR” is any contiguous series ofnucleotide bases that is transcribed, but is not translated. A 5′ UTRlies between the start site of the transcript and the translationinitiation codon and includes the +1 nucleotide. A 3′ UTR lies betweenthe translation termination codon and the end of the transcript. UTRscan have particular functions such as increasing mRNA message stabilityor translation attenuation. Examples of 3′ UTRs include, but are notlimited to polyadenylation signals and transcription terminationsequences.

Variant: The term “variant” is used herein to denote a polypeptide orprotein or polynucleotide molecule that differs from others of its kindin some way. For example, polypeptide and protein variants can consistof changes in amino acid sequence and/or charge and/orpost-translational modifications (such as glycosylation, etc). Likewise,polynucleotide variants can consist of changes that add or delete aspecific UTR or exon sequence. It will be understood that there may besequence variations within sequence or fragments used or disclosed inthis application. Preferably, variants will be such that the sequenceshave at least 80%, preferably at least 90%, 95, 97, 98, or 99% sequenceidentity. Variants preferably measure the primary biological function ofthe native polypeptide or protein or polynucleotide.

2. Introduction

The polynucleotides of the invention comprise promoters and promotercontrol elements that are capable of modulating transcription.

Such promoters and promoter control elements can be used in combinationwith native or heterologous promoter fragments, control elements orother regulatory sequences to modulate transcription and/or translation.

Specifically, promoters and control elements of the invention can beused to modulate transcription of a desired polynucleotide, whichincludes without limitation:

-   (a) antisense;-   (b) ribozymes;-   (c) coding sequences; or-   (d) fragments thereof.    The promoter also can modulate transcription in a host genome in    cis- or in trans-.

In an organism such as a plant, the promoters and promoter controlelements of the instant invention are useful to produce preferentialtranscription which results in a desired pattern of transcript levels inparticular cells, tissues or organs or under particular conditions.

3. Table of Contents

The following description of the present invention is outlined in thefollowing table of contents.

A. Identifying and Isolating Promoter Sequences of the Invention

-   (1) Cloning Methods-   (2) Chemical Synthesis

B. Isolating Related Promoter Sequences

-   (1) Relatives Based on Nucleotide Sequence Identity-   (2) Relatives Based on Coding Sequence Identity-   (3) Relatives Based on Common Function

C. Identifying Control Elements

-   (1) Types of Transcription Control Elements-   (2) Those Described by the Examples-   (3) Those Identifiable by Bioinformatics-   (4) Those Identifiable by In Vitro and In Vivo Assays-   (5) Non-Natural Control Elements

D. Constructing Promoters and Control Elements

-   (1) Combining Promoters and Promoter Control Elements-   (2) Number of Promoter Control Elements-   (3) Spacing Between Control Elements

E. Vectors

-   (1) Modification of Transcription by Promoters and Promoter Control    Elements-   (2) Polynucleotide to be Transcribed-   (3) Other Regulatory Elements-   (4) Other Components of Vectors

F. Insertion of Polynucleotides and Vectors Into a Host Cell

-   (1) Autonomous of the Host Genome-   (2) Integrated into the Host Genome

G. Utility

A. Identifying and Isolating Promoter Sequences of the Invention

The promoters and promoter control elements of the present invention arepresented in the Sequence Listing and were identified from Arabidopsisthaliana or Oryza saliva. Additional promoter sequences encompassed bythe invention can be identified as described below.

(1) Cloning Methods

Isolation from genomic libraries of polynucleotides comprising thesequences of the promoters and promoter control elements of the presentinvention is possible using known techniques.

For example, polymerase chain reaction (PCR) can amplify the desiredpolynucleotides utilizing primers designed from sequences in the rowtitled “Sequences”. Polynucleotide libraries comprising genomicsequences can be constructed according to Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) Ed. (1989) Cold Spring HarborPress, Cold Spring Harbor, N.Y.), for example.

Other procedures for isolating polynucleotides comprising the promotersequences of the invention include, without limitation, tail-PCR and 5′rapid amplification of cDNA ends (RACE). For tail-PCR see, for example,Liu et al. (1995) Plant J 8 (3): 457-463; Liu et al. (1995) Genomics 25:674-681; Liu et al. (1993) Nucl. Acids Res. 21 (14): 3333-3334; and Zoeet al. (1999) BioTechniques 27 (2): 240-248; for RACE see, for example,PCR Protocols: A Guide to Methods and Applications, (1990) AcademicPress, Inc.

(2) Chemical Synthesis

In addition, the promoters and promoter control elements described inthe Sequence Listing can be chemically synthesized according totechniques in common use. See, for example, Beaucage et al. (1981) Tet.Lett. 22: 1859 and U.S. Pat. No. 4,668,777.

Such chemical oligonucleotide synthesis can be carried out usingcommercially available devices, such as Biosearch 4600 or 8600 DNAsynthesizer by Applied Biosystems, a division of Perkin-Elmer Corp.(Foster City, Calif., USA) and Expedite by Perceptive Biosystems(Framingham, Mass., USA).

Synthetic RNA, including natural and/or analog building blocks, can besynthesized on the Biosearch 8600 machines (see above).

Oligonucleotides can be synthesized and then ligated together toconstruct the desired polynucleotide.

B. Isolating Related Promoter Sequences

Included in the present invention are promoter and promoter controlelements that are related to those described in the Sequence Listing.Such related sequences can be isolated using

-   (a) nucleotide sequence identity,-   (b) coding sequence identity or-   (c) common function or gene products.    Relatives can include both naturally occurring promoters and    non-natural promoter sequences. Non-natural related promoters    include nucleotide substitutions, insertions or deletions of    naturally-occurring promoter sequences that do not substantially    affect transcription modulation activity. For example, the binding    of relevant DNA binding proteins can still occur with the    non-natural promoter sequences and promoter control elements of the    present invention.

According to current knowledge, promoter sequences and promoter controlelements exist as functionally important regions, such as proteinbinding sites and spacer regions. These spacer regions are apparentlyrequired for proper positioning of the protein binding sites. Thus,nucleotide substitutions, insertions and deletions can be tolerated inthe spacer regions to a certain degree without loss of function.

In contrast, less variation is permissible in the functionally importantregions since changes in the sequence can interfere with proteinbinding. Nonetheless, some variation in the functionally importantregions is permissible so long as function is conserved.

The effects of substitutions, insertions and deletions to the promotersequences or promoter control elements may be to increase or decreasethe binding of relevant DNA binding proteins to modulate transcriptlevels of a polynucleotide to be transcribed. Effects may includetissue-specific or condition-specific modulation of transcript levels ofthe polypeptide to be transcribed. Polynucleotides representing changesto the nucleotide sequence of the DNA-protein contact region byinsertion of additional nucleotides, changes to identity of relevantnucleotides, including use of chemically-modified bases, or deletion ofone or more nucleotides are considered encompassed by the presentinvention.

(1) Relatives Based on Nucleotide Sequence Identity

Included in the present invention are promoters exhibiting nucleotidesequence identity to those described in the Sequence Listing.

Definition

Typically, such related promoters exhibit at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95%, even more preferably, at least 96%, 97%,98% or 99% sequence identity compared to those shown in the SequenceListing. Such sequence identity can be calculated by the algorithms andcomputers programs described above.

Usually, such sequence identity is exhibited in an alignment region thatis at least 75% of the length of a sequence shown in the SequenceListing or corresponding full-length sequence; more usually at least80%; more usually, at least 85%, more usually at least 90%, and mostusually at least 95%, yet even more usually, at least 96%, 97%, 98% or99% of the length of a sequence shown in the Sequence Listing.

The percentage of the alignment length is calculated by counting thenumber of bases of the sequence in the region of strongest alignment,e.g. a continuous region of the sequence that contains the greatestnumber of bases that are identical to the bases between two sequencesthat are being aligned. The number of bases in the region of strongestalignment is divided by the total base length of a sequence in theSequence Listing.

These related promoters generally exhibit similar preferentialtranscription as those promoters described in the Sequence Listing andas described in the “observed expression pattern” and “expectedexpression pattern” fields of the reports of Table 1.

Construction of Polynucleotides

Naturally occurring promoters that exhibit nucleotide sequence identityto those shown in the Sequence Listing can be isolated using thetechniques as described above. More specifically, such related promoterscan be identified, for example, with typical hybridization proceduressuch as Southern blots or probing of polynucleotide libraries usingvarying stringencies (see above).

Non-natural promoter variants of those shown in the Sequence Listing canbe constructed using cloning methods that incorporate the desirednucleotide variation. For example see Ho et al. (1989) Gene 77:51-59,which describes a site directed mutagenesis procedure using PCR.

Any related promoter showing sequence identity to those shown in theSequence Listing can be chemically synthesized as described above.

Also, the present invention includes non-natural promoters that exhibitthe above-sequence identity to those in the Sequence Listing.

The promoters and promoter control elements of the present invention mayalso be synthesized with 5′ or 3′ extensions to facilitate additionalmanipulation, for instance.

Testing of Polynucleotides

Polynucleotides of the invention were tested for activity by cloning thesequence into an appropriate vector, transforming plants with theconstruct and assaying for marker gene expression. Recombinant DNAconstructs were prepared which comprise the polynucleotide sequences ofthe invention inserted into a vector suitable for transformation ofplant cells. The construct can be made using standard recombinant DNAtechniques (Sambrook et al. 1989) and can be introduced to the speciesof interest by Agrobacterium-mediated transformation or by other meansof transformation as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by

-   (a) BAC: Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA 89:    8794-8797; Hamilton et al. (1996) Proc. Natl. Acad. Sci. USA 93:    9975-9979;-   (b) YAC: Burke et al. (1987) Science 236:806-812;-   (c) PAC: Sternberg N. et al. (1990) Proc Natl Acad Sci USA. 87    (1):103-7;-   (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al. (1995) Nucl    Acids Res 23: 4850-4856;-   (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et    al. (1983) J. Mol Biol 170: 827-842; or Insertion vector, e.g.,    Huynh et al., In: Glover NM (ed) DNA Cloning: A practical Approach,    Vol. 1 Oxford: IRL Press (1985); T-DNA gene fusion vectors: Walden    et al. (1990) Mol Cell Biol 1: 175-194; and-   (g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a sequence of thepresent invention operationally linked to any marker gene. Thepolynucleotide was identified as a promoter by the expression of themarker gene. Although many marker genes can be used, Green FluorescentProtein (GFP) is preferred. The vector may also comprise a marker genethat confers a selectable phenotype on plant cells. The marker mayencode biocide resistance, particularly antibiotic resistance, such asresistance to kanamycin, G418, bleomycin, hygromycin or herbicideresistance, such as resistance to chlorosulfuron or phosphinotricin.Vectors can also include origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, introns, etc.

Promoter Control Elements of the Invention

The promoter control elements of the present invention include thosethat comprise a sequence shown in the Sequence Listing or fragmentsthereof. The size of the fragments can range from 5 bases to 10kilobases (kb). Typically, the fragment size is no smaller than 8 bases;more typically, no smaller than 12; more typically, no smaller than 15bases; more typically, no smaller than 20 bases; more typically, nosmaller than 25 bases; even more typically, no more than 30, 35, 40 or50 bases.

Usually, the fragment size is no larger than 5 kb bases; more usually,no larger than 2 kb; more usually, no larger than 1 kb; more usually, nolarger than 800 bases; more usually, no larger than 500 bases; even moreusually, no more than 250, 200, 150 or 100 bases.

Relatives Based on Nucleotide Sequence Identity

Included in the present invention are promoter control elementsexhibiting nucleotide sequence identity to those described in theSequence Listing or fragments thereof.

Typically, such related promoters exhibit at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95%, even more preferably, at least 96%, 97%,98% or 99% sequence identity compared to those shown in the SequenceListing. Such sequence identity can be calculated by the algorithms andcomputers programs described above.

Promoter Control Element Configuration

A common configuration of the promoter control elements in RNApolymerase II promoters is described in “Models for prediction andrecognition of eukaryotic promoters”, T. Werner (1999) Mammalian Genome10, 168-175.

Promoters are generally modular in nature. Promoters can consist of abasal promoter which functions as a site for assembly of a transcriptioncomplex comprising an RNA polymerase, for example RNA polymerase II. Atypical transcription complex will include additional factors such asTF_(II)B, TF_(II)D and TF_(II)E. Of these, TF_(II)D appears to be theonly one to bind DNA directly. The promoter might also contain one ormore promoter control elements such as the elements discussed above.These additional control elements may function as binding sites foradditional transcription factors that have the function of modulatingthe level of transcription with respect to tissue specificity, oftranscriptional responses to particular environmental or nutritionalfactors and the like.

One type of promoter control element is a polynucleotide sequencerepresenting a binding site for proteins. Typically, within a particularfunctional module, protein binding sites constitute regions of 5 to 60,preferably 10 to 30, more preferably 10 to 20 nucleotides. Within suchbinding sites, there are typically 2 to 6 nucleotides which specificallycontact amino acids of the nucleic acid binding protein.

The protein binding sites are usually separated from each other by 10 toseveral hundred nucleotides, typically by 15 to 150 nucleotides, oftenby 20 to 50 nucleotides.

Further, protein binding sites in promoter control elements oftendisplay dyad symmetry in their sequence. Such elements can bind severaldifferent proteins and/or a plurality of sites can bind the sameprotein. Both types of elements may be combined in a region of 50 to1,000 base pairs.

Binding sites for any specific factor have been known to occur almostanywhere in a promoter. For example, functional AP-1 binding sites canbe located far upstream, as in the rat bone sialoprotein gene where anAP-1 site located about 900 nucleotides upstream of the transcriptionstart site suppresses expression (Yamauchi et al. (1996) Matrix Biol.15, 119-130). Alternatively, an AP-1 site located close to thetranscription start site plays an important role in the expression ofMoloney murine leukemia virus (Sap et al. (1989) Nature 340, 242-244.

(2) Those Identifiable by Bioinformatics

Promoter control elements from the promoters of the instant inventioncan be identified utilizing bioinformatic or computer driven techniques.

One method uses a computer program AlignACE to identify regulatorymotifs in genes that exhibit common preferential transcription across anumber of time points. The program identifies common sequence motifs insuch genes. See, Roth et al. (1998) Nature Biotechnol. 16: 949-945;Tavazoie et al. (1999) Nat Genet Jul; 22 (3):281-5;

Genomatix also makes available a GEMS Launcher program and otherprograms to identify promoter control elements and configuration of suchelements. Genomatix is located in Munich, Germany.

Other references also describe detection of promoter modules by modelsindependent of overall nucleotide sequence similarity. See, forinstance, Klingenhoff et al. (1999) Bioinformatics 15: 180-186.

Protein binding sites of promoters can be identified as reported in“Computer-assisted prediction, classification, and delimination ofprotein binding sites in nucleic acids”, Frech et al. (1993) NucleicAcids Research 21 (7): 1655-1664.

Other programs used to identify protein binding sites include, forexample, Signal Scan (Prestridge et al. (1996) Comput. Appl. Biosci. 12:157-160); Matrix Search (Chen et al. (1995) Comput. Appl. Biosci. 11:563-566), available as part of Signal Scan 4.0; MatInspector (Ghosh etal. (1993) Nucl. Acid Res. 21: 3117-3118) available via the internet;ConsInspector (Frech et al. (1993) Nucl. Acids Res. 21: 1655-1664),available via the internet; TFSearch and TESS.

Frech et al. (1997) “Software for the analysis of DNA sequence elementsof transcription”, Bioinformatics & Sequence Analysis, Vol. 13, no. 1,89-97 is a review of different software for analysis of promoter controlelements. This paper also reports the usefulness of matrix-basedapproaches to yield more specific results.

For other procedures, see Fickett et al. (2000) Curr. Op. Biotechnol.11: 19-24 and Quandt et al. (1995) Nucleic Acids Res. 23: 4878-4884.

(3) Those Identifiable by In-Vitro and In-Vivo Assays

Promoter control elements can also be identified with in-vitro assayssuch as transcription detection methods and with in-vivo assays such asenhancer trapping protocols.

In-Vitro Assays

Examples of in-vitro assays include detection of binding of proteinfactors that bind promoter control elements. Fragments of the instantpromoters can be used to identify the location of promoter controlelements. Another option for obtaining a promoter control element withdesired properties is to modify known promoter sequences. This is basedon the fact that the function of a promoter is dependent on theinterplay of regulatory proteins that bind to specific, discretenucleotide sequences (“motifs”) in the promoter. Such interplaysubsequently affects the general transcription machinery and regulatestranscription efficiency. These regulatory proteins are positiveregulators or negative regulators (repressors) and one protein can havea dual role depending on the context (Johnson and McKnight, (1989) Annu.Rev. Biochem. 58:799-839).

One type of in-vitro assay uses a known DNA binding factor to isolateDNA fragments that bind. If a fragment or promoter variant does notbind, then a promoter control element has been removed or disrupted. Forspecific assays, see, for instance, Luo et al. (1997) J. Mol. Biol.266:470, Chusacultanachai et al. (1999) J. Biol. Chem. 274:23591, Fabbroet al. (1995) Biochem. Biophys. Res. Comm. 213:781).

Alternatively, a DNA fragment suspected of conferring a particularpattern of expression can be examined for the ability to bindtranscription factors responsible for generating the particular patternusing methods such as DNA footprinting (e.g. Cousins et al. (2000)Immunology 99:101 and V. Kolla et al. (1999) Biochem. Biophys. Res.Comm. 266:5) or “mobility-shift” assays (Fabiani et al. (2000) J.Biochem. 347:147 and Sugiura et al. (2000) J. Biochem 347:155) orfluorescence polarization (e.g. Royer et al. U.S. Pat. No. 5,445,935).Both mobility shift and DNA footprinting assays can also be used toidentify portions of large DNA fragments that are bound by proteins inunpurified transcription extracts prepared from tissues or organs ofinterest.

Cell-free transcription extracts can be prepared and used to directlyassay in a reconstitutable system (Narayan et al. (2000) Biochemistry39:818).

In-Vivo Assays

Promoter control elements can be identified with reporter genes inin-vivo assays with the use of fragments of the instant promoter,polynucleotides or variants thereof. That is, a fragment(s) comprising abasal or “core” promoter operably linked to a reporter sequence can beinserted into a vector. When transcribed, a detectable label isproduced. Examples of reporter genes include those encoding luciferase,green fluorescent protein, GUS, neo, cat and bar. Alternatively, thetranscribed reporter sequence can be detected with AFLP and microarraytechniques.

In promoter probe vector systems, genomic DNA fragments are insertedupstream of the coding sequence of a reporter gene which is expressedonly when the cloned fragment contains DNA having transcriptionmodulation activity (Neveet al. (1979) Nature 277:324-325). Notranscription occurs when control elements are present in the fragmentor when control elements present are disrupted. Probe vectors have beendesigned for assaying transcription modulation in E. coli (An et al.(1979) J. Bact. 140:400-407) and other bacterial hosts (Band et al.(1983) Gene 26:313-315 and Achen (1986) Gene 45:45-49), yeast (Goodey etal. (1986) Mol. Gen. Genet. 204:505-511) and mammalian cells (Pater etal. (1984) J. Mol. App. Gen. 2:363-371).

A different design of a promoter/control element trap includes packaginginto retroviruses for more efficient delivery into cells. One type ofretroviral enhancer trap was described by von Melchner et al. (GenesDev. (1992); U.S. Pat. No. 5,364,783). The basic design of this vectorincludes a reporter protein coding sequence engineered into the U3portion of the 3′ LTR. No splice acceptor consensus sequences areincluded, limiting its utility to work as an enhancer trap only. Adifferent approach to a gene trap using retroviral vectors was pursuedby Friedrich and Soriano (Genes Dev. 1991) who engineered a lacZ-neofusion protein linked to a splicing acceptor. LacZ-neo fusion proteinexpression from trapped loci allows not only for drug selection, butalso for visualization of 13-galatactosidase expression using thechromogenic substrate, X-gal.

A general review of tools for identifying transcriptional regulatoryregions of genomic DNA is provided by J. W. Fickett et al. (Curr. Opn.Biotechnol (2000) 11:19).

(4) Non-Natural Control Elements

Non-natural control elements can be constructed by inserting, deletingor substituting nucleotides into the promoter control elements describedabove. Such control elements are capable of transcription modulationthat can be determined using any of the assays described above.

C. Constructing Promoters with Control Elements

(1) Combining Promoters and Promoter Control Elements

The promoter polynucleotides and promoter control elements of thepresent invention, both naturally occurring and synthetic, can becombined with each other to produce the desired preferentialtranscription. In addition, the polynucleotides of the invention can becombined with other known sequences to generate promoters useful formodulating, for example, tissue-specific transcription orcondition-specific transcription. Such preferential transcription can bedetermined using the techniques or assays described above.

The relatives, fragments and variants as well as full-length sequencesshown in the Sequence Listing are useful alone or in combination.

The location and relation of promoter control elements within a promotercan affect the ability of the promoter to modulate transcription. Theorder and spacing of control elements is a factor when constructingpromoters.

(2) Number of Promoter Control Elements

Promoters can contain any number of control elements. For example, apromoter can contain multiple transcription binding sites or othercontrol elements. One element may confer tissue or organ specificity,another element may limit transcription to specific time periods, etc.Typically, promoters will contain at least a basal or core promoter asdescribed above. Any additional element can be included as desired. Forexample, a fragment comprising a basal or “core” promoter can be fusedwith another fragment with any number of additional control elements.

(3) Spacing Between Control Elements

Spacing between control elements or the configuration or controlelements can be determined or optimized to permit the desiredpolynucleotide or protein-polynucleotide interactions to occur.

For example, if two transcription factors bind to a promotersimultaneously or relatively close in time, the binding sites are spacedto allow each factor to bind without steric hinderance. The spacingbetween two such hybridizing control elements can be as small as aprofile of a protein bound to a control element. In some cases, twoprotein binding sites can be adjacent to each other when the proteinsbind at different times during the transcription process.

Further, when two control elements hybridize the spacing between suchelements will be sufficient to allow the promoter polynucleotide to forma hairpin or loop so as to permit the two elements to bind. The spacingbetween two such hybridizing control elements can be as small as a t-RNAloop, to as large as 10 kb.

Typically, the spacing is no smaller than 5 bases, more typically nosmaller than 8, more typically no smaller than 15 bases, more typicallyno smaller than 20 bases, more typically no smaller than 25 bases, evenmore typically no more than 30, 35, 40 or 50 bases.

Usually, the fragment size in no larger than 5 kb bases, more usually nolarger than 2 kb, more usually no larger than 1 kb, more usually nolarger than 800 bases, more usually no larger than 500 bases, even moreusually no more than 250, 200, 150 or 100 bases.

Such spacing between promoter control elements can be determined usingthe techniques and assays described above.

(4) Other Promoters

The following are promoters that are induced under stress conditions andcan be combined with those of the present invention: 1dh1 (oxygenstress, tomato see Germain and Ricard (1997) Plant Mol Biol 35:949-54),GPx and CAT (oxygen stress, mouse, see Franco et al. (1999) Free RadicBiol Med 27:1122-32), ci7 (cold stress, potato, see Kirch et al. (1997)Plant Mol Biol. 33:897-909), Bz2 (heavy metals, maize, see Mans andWalbot (1997) Plant Physiol 113:93-102), HSP32 (hyperthermia, rat, seeRaju and Maines (1994) Biochim Biophys Acta 1217:273-80); MAPKAPK-2(heat shock, Drosophila, see Larochelle and Suter (1995) Gene163:209-14).

In addition, the following promoters are examples those induced by thepresence or absence of light and can be used in combination with thoseof the present invention: Topoisomerase II (pea, see Reddy et al. (1999)Plant Mol Biol 41:125-37), chalcone synthase (soybean, see Wingender etal. (1989) Mol Gen Genet 218:315-22), mdm2 gene (human tumor, seeSaucedo et al. (1998) Cell Growth Differ 9:119-30), Clock and BMAL1(rat, see Namihira et al. (1999) Neurosci Lett 271:1-4), PHYA(Arabidopsis, see Canton and Quail 1999 Plant Physiol 121:1207-16),PRB-1b (tobacco, see Sessa et al. (1995) Plant Mol Biol 28:537-47) andYpr10 (common bean, see Walter et al. (1996) Eur J Biochem 239:281-93).

The promoters and control elements of the following genes can be used incombination with the present invention to confer tissue specificity: forroots MipB (iceplant, Yamada et al. (1995) Plant Cell 7:1129-42) andSUCS (root nodules, broadbean, Kuster et al. (1993) Mol Plant MicrobeInteract 6:507-14), for leaves OsSUT1 (rice, Hirose et al. (1997) PlantCell Physiol 38:1389-96), for siliques Msg (soybean, Stomvik et al.(1999) Plant Mol Biol 41:217-31) and for inflorescence (Arabidopsis,Shani et al. (1997) Plant Mol Biol 34 (6):837-42) and ACT11(Arabidopsis, Huang et al. (1997) Plant Mol Biol 33:125-39).

Still other promoters are affected by hormones or participate inspecific physiological processes, which can be used in combination withthose of present invention. Some examples are the ACC synthase gene thatis induced differently by ethylene and brassinosteroids (mung bean, Yiet al. (1999) Plant Mol Biol 41:443-54), the TAPG1 gene that is activeduring abscission (tomato, Kalaitzis et al. (1995) Plant Mol Biol28:647-56) and the 1-aminocyclopropane-1-carboxylate synthase gene(carnation, Jones et al. (1995) Plant Mol Biol 28:505-12) and theCP-2/cathepsin L gene (rat, Kim and Wright (1997) Biol Reprod57:1467-77), which are both active during senescence.

E. Vectors

Vectors are a useful component of the present invention. In particular,vectors can deliver the present promoters and/or promoter controlelements to a cell. For the purposes of this invention, such deliveryranges from randomly introducing the promoter or promoter controlelement alone into a cell to integrating the vector containing thepromoter or promoter control element into a cell's genome. Thus, avector need not be limited to a DNA molecule such as a plasmid, cosmidor bacterial phage that has the capability of replicating autonomouslyin a host cell. All other manner of delivery of the promoters andpromoter control elements of the invention are envisioned. The variousT-DNA vector types are preferred vectors for use with the presentinvention. Many useful vectors are commercially available.

It may also be useful to attach a marker sequence to the presentpromoter and promoter control element in order to determine activity ofsuch sequences. Marker sequences typically include genes that provideantibiotic resistance, such as tetracycline resistance, hygromycinresistance or ampicillin resistance, or provide herbicide resistance.Specific selectable marker genes may be used to confer resistance toherbicides such as glyphosate, glufosinate or broxynil (Comai et al.(1985) Nature 317: 741-744; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; and Stalker et al. (1988) Science 242: 419-423). Other markergenes exist which provide hormone responsiveness.

(1) Modification of Transcription by Promoters and Promoter ControlElements

The promoter or promoter control element of the present invention may beoperably linked to a polynucleotide to be transcribed. In this manner,the promoter or promoter control element modifys transcription bymodulating transcript levels of that polynucleotide when inserted into agenome.

The promoter or promoter control element need not be linked, operably orotherwise, to a polynucleotide to be transcribed before being insertedinto a genome. For example, the promoter or promoter control element canbe inserted into the genome in front of a polynucleotide already presenttherein. Here, the promoter or promoter control element modulates thetranscription of a polynucleotide that was already present in thegenome. This polynucleotide may be native to the genome or inserted atan earlier time.

Alternatively, the promoter or promoter control element can simply beinserted into a genome or maintained extrachromosomally as a way todivert the transcription resources of the system to itself. See, forexample, Vaucheret et al. (1998) Plant J 16: 651-659. This approach maybe used to downregulate the transcript levels of a group ofpolynucleotide(s).

(2) Polynucleotide to be Transcribed

The nature of the polynucleotide to be transcribed is not limited.Specifically, the polynucleotide may include sequences that will haveactivity as RNA as well as sequences that result in a polypeptideproduct. These sequences may include, but are not limited to antisensesequences, ribozyme sequences, spliceosomes, amino acid coding sequencesand fragments thereof.

Specific coding sequences may include, but are not limited to endogenousproteins or fragments thereof, or heterologous proteins including markergenes or fragments thereof.

Promoters and control elements of the present invention are useful formodulating metabolic or catabolic processes. Such processes include, butare not limited to secondary product metabolism, amino acid synthesis,seed protein storage, oil development, pest defense and nitrogen usage.Some examples of genes, transcripts, peptides or polypeptidesparticipating in these processes which can be modulated by the presentinvention: are tryptophan decarboxylase (tdc), strictosidine synthase(str1), dihydrodipicolinate synthase (DHDPS), aspartate kinase (AK), 2Salbumin, alpha-, beta-, and gamma-zeins, ricinoleate, 3-ketoacyl-ACPsynthase (KAS), Bacillus thuringiensis (Bt) insecticidal protein, cowpeatrypsin inhibitor (CpTI), asparagine synthetase and nitrite reductase.Alternatively, expression constructs can be used to inhibit expressionof these peptides and polypeptides by incorporating the promoters inconstructs for antisense use, co-suppression use or for the productionof dominant negative mutations.

(3) Other Regulatory Elements

As explained above, several types of regulatory elements existconcerning transcription regulation. Each of these regulatory elementsmay be combined with the present vector if desired.

(4) Other Components of Vectors

Translation of eukaryotic mRNA is often initiated at the codon thatencodes the first methionine. Thus, when constructing a recombinantpolynucleotide for expressing a protein product according to the presentinvention, it is preferable to ensure that no intervening codonsencoding a methionine are contained within the linkage between thepolynucleotide to be transcribed, or a functional derivative thereof,and the 3′ portion of the promoter, preferably including the TATA box.

The vector of the present invention may contain additional components.For example, an origin of replication that allows for replication of thevector in a host cell may be added. In addition, homologous sequencesflanking a target location in the genome may be added to allow forsite-specific recombination of a specific sequence contained in thevector. T-DNA sequences also allow for insertion of a specific sequencerandomly into a target genome, but in a random manner.

The vector may also contain a plurality of restriction sites forinsertion of the promoter and/or promoter control elements of thepresent invention as well as any polynucleotide to be transcribed. Thevector can additionally contain selectable marker genes. The vector canalso contain a transcriptional and translational initiation regionand/or a transcriptional and translational termination region thatfunctions in the host cell. The termination region may be native withthe transcriptional initiation region, may be native with thepolynucleotide to be transcribed or may be derived from another source.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the polynucleotide to be transcribed may be optimizedfor increased expression in a certain host cell. For example, thepolynucleotide can be synthesized using preferred codons for improvedtranscription and translation. See U.S. Pat. Nos. 5,380,831, 5,436, 391and Murray et al. (1989) Nucleic Acids Res. 17:477-498.

Additional sequence modifications include elimination of sequencesencoding spurious polyadenylation signals, exon intron splice sitesignals, transposon-like repeats and other such sequences wellcharacterized as deleterious to expression. The G-C content of thepolynucleotide may be adjusted to the average levels for a givencellular host, as calculated by reference to known genes expressed inthe host cell. The polynucleotide sequence may be modified to avoidhairpin secondary mRNA structures.

A general description of expression vectors and reporter genes can befound in Gruber et al., “Vectors for Plant Transformation”, in Methodsin Plant Molecular Biology & Biotechnology (1993) Glich et al. eds., pp.89-119, CRC Press). Moreover GUS expression vectors and GUS genecassettes are available from Clonetech Laboratories, Inc. (Palo Alto,Calif.) while luciferase expression vectors and luciferase genecassettes are available from Promega Corp. (Madison, Wis.). GFP vectorsare available from Aurora Biosciences.

F. Polynucleotide Insertion Into a Host Cell

The polynucleotides according to the present invention can be insertedinto a host cell. A host cell includes but is not limited to a plant,mammalian, insect, yeast and prokaryotic cell, preferably a plant cell.

The method of insertion into the host cell genome is chosen based onconvenience. For example, the insertion into the host cell genome mayeither be accomplished by vectors that integrate into the host cellgenome or by vectors which exist independent of the host cell genome.

(1) Polynucleotides Autonomous of the Host Genome

The polynucleotides of the present invention can exist autonomously orindependent of the host cell genome. Vectors of these types are known inthe art and include, for example, certain types of non-integrating viralvectors, autonomously replicating plasmids, artificial chromosomes andthe like.

Additionally, in some cases transient expression of a polynucleotide maybe desired.

(2) Polynucleotides Integrated into the Host Genome

The promoter sequences, promoter control elements or vectors of thepresent invention can be transformed into host cells. Thesetransformations can be into protoplasts or isolated cells or intacttissues. Preferably, expression vectors are introduced into intacttissue. General methods of culturing plant tissues are provided forexample by Maki et al. (“Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology & Biotechnology (1993)Glich et al. eds., pp. 67-88 CRC Press) and by Phillips et al.“Cell-Tissue Culture and In-Vitro Manipulation” in Corn & CornImprovement, 3rd Edition 10Sprague et al. (1998) eds. pp. 345-387)American Society of Agronomy Inc. et al.

Methods of introducing polynucleotides into plant tissue include thedirect infection or co-cultivation of a plant cell with Agrobacteriumtumefaciens (Horsch et al. (1985) Science 227:1229). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al. supra.

Alternatively, polynucleotides are introduced into plant cells or otherplant tissues using a direct gene transfer method such asmicroprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably, polynucleotides are introduced into planttissues using the microprojectile media delivery with the biolisticdevice. See, for example, Tomes et al., “Direct DNA transfer into intactplant cells via microprojectile bombardment” In: Plant Cell, Tissue andOrgan Culture: Fundamental Methods (1995) Gamborg and Phillips eds.Springer Verlag, Berlin.

In another embodiment of the current invention, expression constructscan be used for gene expression in callus culture for the purpose ofexpressing marker genes encoding peptides or polypeptides that allowidentification of transformed plants. Here, a promoter that isoperatively linked to a polynucleotide to be transcribed is transformedinto plant cells and the transformed tissue is then placed oncallus-inducing media. If the transformation is conducted with leafdiscs, for example, callus will initiate along the cut edges. Oncecallus growth has initiated, callus cells can be transferred to callusshoot-inducing or callus root-inducing media. Gene expression will occurin the callus cells developing on the appropriate media: callusroot-inducing promoters will be activated on callus root-inducing media,etc. Examples of such peptides or polypeptides useful as transformationmarkers include, but are not limited to barstar, glyphosate,chloramphenicol acetyltransferase (CAT), kanamycin, spectinomycin,streptomycin or other antibiotic resistance enzymes, green fluorescentprotein (GFP) and β-glucuronidase (GUS), etc. Some of the promoters inthe Sequence Listing will also be capable of sustaining expression insome tissues or organs after the initiation or completion ofregeneration. Examples of these tissues or organs are somatic embryos,cotyledon, hypocotyl, epicotyl, leaf, stems, roots, flowers and seed.

Integration into the host cell genome also can be accomplished bymethods known in the art such as by homologous sequences or T-DNAdiscussed above or by using the cre-lox system (Vergunst et al. (1998)Plant Mol. Biol. 38:393).

G. Utility

Common Uses

In yet another embodiment, the promoters of the present invention can beused to further understand developmental mechanisms. For example,promoters that are specifically induced during callus formation, somaticembryo formation, shoot formation or root formation can be used toexplore the effects of overexpression, repression or ectopic expressionof target genes, or for isolation of trans-acting factors.

The vectors of the invention can be used not only for expression ofcoding regions but may also be used in exon-trap cloning, or promotertrap procedures to detect differential gene expression in varioustissues. See Lindsey et al (1993) “Tagging Genomic Sequences That DirectTransgene Expression by Activation of a Promoter Trap in Plants,”Transgenic Research 2:3347 and Auch et al. “Exon Trap Cloning: Using PCRto Rapidly Detect and Clone Exons from Genomic DNA Fragments,” NucleicAcids Research, 18:674.

Entrapment vectors, first described for use in bacteria (Casadaban andCohen (1979) Proc. Nat. Aca. Sci. U.S.A. 76: 4530 and Casadaban et al.(1980) J. Bacteriol. 143: 971) permit selection of insertional eventsthat lie within coding sequences. Entrapment vectors can be introducedinto pluripotent ES cells in culture and then passed into the germlinevia chimeras (Gossler et al. (1989) Science 244: 463 and Skarnes (1990)Biotechnology 8: 827). Promoter or gene trap vectors often contain areporter gene, e.g. lacZ, lacking its own promoter and/or spliceacceptor sequence upstream. That is, promoter gene traps contain areporter gene with a splice site but no promoter. If the vector lands ina gene and is spliced into the gene product, then the reporter gene isexpressed.

Recently, the isolation of preferentially-induced genes has been madepossible with the use of sophisticated promoter traps (e.g. IVET) thatare based on conditional auxotrophy complementation or drug resistance.In one IVET approach, various bacterial genome fragments are placed infront of a necessary metabolic gene that is coupled to a reporter gene.The DNA constructs are inserted into a bacterial strain otherwiselacking the metabolic gene and the resulting bacteria are used to infectthe host organism. Only bacteria expressing the metabolic gene survivein the host organism. Consequently, inactive constructs can beeliminated by harvesting only bacteria that survive for some minimumperiod in the host. At the same time, constitutively active constructscan be eliminated by selecting only bacteria that do not express thereporter gene under laboratory conditions. The bacteria selected by sucha method contain constructs that are selectively induced only duringinfection of the host. The IVET approach can be modified in plants toidentify genes induced in either the bacteria or the plant cells uponpathogen infection or root colonization. For information on IVET see thefollowing articles: Mahan et al. (1993) Science 259:686-688, Mahan etal. (1995) PNAS USA 92:669-673, Heithoff et al. (1997) PNAS USA94:934-939, and Wang et al. (1996) PNAS USA. 93:10434.

Constitutive Transcription

Promoters and control elements providing constitutive transcription aredesired for modulation of transcription in most cells of an organismunder most environmental conditions. In a plant, for example,constitutive transcription is useful for modulating genes involved indefense, pest resistance, herbicide resistance, etc.

Constitutive up-regulation and down-regulation of transcription areuseful for these applications. For instance, genes, transcripts and/orpolypeptides that increase defense, pest and herbicide resistance mayrequire constitutive up-regulation of transcription. In contrast,constitutive down-regulation of transcriptional may be desired toinhibit those genes, transcripts, and/or polypeptides that lowerdefense, pest and herbicide resistance.

Typically, promoter or control elements that provide constitutivetranscription produce transcription levels that are statisticallysimilar in many tissues and environmental conditions observed.

Calculation of P-value from the different observed transcript levels isone means of determining whether a promoter or control element isproviding constitutive up-regulation. P-value is the probability thatthe difference of transcript levels is not statistically significant.The higher the P-value, the more likely the difference of transcriptlevels is not significant. One formula used to calculate P-value is asfollows:

∫ϕ(x)x, integrated  from  a  to  ∞, where  ϕ(x)  is  a  normal  distribution;${{where}\mspace{14mu} a} = \frac{{{Sx} - \mu}}{{\sigma ( {{all}\mspace{14mu} {Samples}\mspace{14mu} {except}\mspace{14mu} {Sx}} )};}$where  Sx = the  intensity  of  the  sample  of  interest${{{where}\mspace{14mu} \mu} = {{is}\mspace{14mu} {the}\mspace{14mu} {average}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {intensities}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {samples}\mspace{14mu} {except}\mspace{14mu} {Sx}}},{= \frac{( {\Sigma \; S\; 1\mspace{14mu} \ldots \mspace{20mu} {Sn}} ) - {Sx}}{n - 1}}$

where σ (S1 . . . S11, not including Sx)=the standard deviation of allsample intensities except Sx.

The P-value from the formula ranges from 1.0 to 0.0.

Usually, each P-value of the transcript levels produced by the promoteror control element and observed in a majority of cells, tissues ororgans under various environmental conditions is greater than 10⁻⁸; moreusually, greater than 10⁻⁷; even more usually, greater than 10⁻⁶; evenmore usually, greater than 10⁻⁵ or 10⁻⁴.

For up-regulation of transcription, promoter and control elementsproduce transcript levels that are above background of the assay.

Stress Induced Preferential Transcription

Promoters and control elements providing modulation of transcriptionunder oxidative, drought, oxygen, wound and methyl jasmonate stress areparticularly useful for producing host cells or organisms that are moreresistant to biotic and abiotic stresses. For example, in a plantmodulation of genes, transcripts and/or polypeptides in response tooxidative stress can protect cells against damage caused by oxidativeagents such as hydrogen peroxide and other free radicals.

Drought induction of genes, transcripts and/or polypeptides are usefulto increase the viability of a plant, for example when water is alimiting factor. In contrast, genes, transcripts and/or polypeptidesinduced during oxygen stress can help the flood tolerance of a plant.

The promoters and control elements of the present invention can modulatethe plant's response to stresses. Examples of some genes involved instress condition responses are VuPLD1 (drought stress, Cowpea; Pham-Thiet al. (1999) Plant Mol. Biol 1257-65), pyruvate decarboxylase (oxygenstress, rice; Rivosal et al. (1997) Plant Physiol. 114 (3): 1021-29),and the chromoplast specific carotenoid gene (oxidative stress,Capsicum; see Bouvier et al. (1998) J Biol Chem 273: 30651-59).

Promoters and control elements providing preferential transcriptionduring wounding or that are induced by methyl jasmonate can produce adefense response in host cells or organisms. In a plan, preferentialmodulation of genes, transcripts and/or polypeptides under suchconditions is useful to induce a defense response to mechanicalwounding, pest or pathogen attack or treatment with certain chemicals.

Promoters and control elements of the present invention also can triggera response similar to those described for cf9 (viral pathogen, tomato;O'Donnell et al. (1998) Plant J 14 (1): 137-42), hepatocyte growthfactor activator inhibitor type 1 (HAI-1), which enhances tissueregeneration (tissue injury, human; Koono et al. (1999) J HistochemCytochem 47: 673-82), copper amine oxidase (CuAO) induced duringontogenesis and wound healing (wounding, chick-pea; Rea et al. (1998)FEBS Letters 437: 177-82), proteinase inhibitor II (wounding, potato;Pena-Cortes et al. (1988) Planta 174: 84-89), protease inhibitor II(methyl jasmonate, tomato; Farmer and Ryan (1990) Proc Natl Acad Sci USA87: 7713-7716) and two vegetative storage protein genes VspA and VspB(wounding, jasmonic acid and water deficit; soybean; Mason and Mullet(1990) Plant Cell 2: 569-579).

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease oxidative, flood or drought tolerance may require up-regulationof transcription. In contrast, transcriptional down-regulation may bedesired to inhibit those genes, transcripts and/or polypeptides thatlower such tolerance.

Typically, promoter or control elements which provide preferentialtranscription in wounding or under methyl jasmonate induction producetranscript levels that are statistically significantly altered ascompared to cell types, organs or tissues under other conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Light Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by light exposure can be utilized to modulate growth, metabolismand development to increase drought tolerance and to decrease damagefrom light stress for host cells or organisms. In a plant, modulation ofgenes, transcripts and/or polypeptides in response to light is useful

-   (1) to increase the photosynthetic rate;-   (2) to increase storage of certain molecules in leaves or green    parts only, e.g. silage with high protein or starch content;-   (3) to modulate production of exogenous compositions in green    tissue, e.g. certain feed enzymes;-   (4) to induce growth or development, such as fruit development and    maturity, during extended exposure to light;-   (5) to modulate guard cells to control the size of stomata in leaves    to prevent water loss, or-   (6) to induce accumulation of beta-carotene to help plants cope with    light induced stress.    The promoters and control elements of the present invention can also    trigger responses similar to those described for: abscisic acid    insensitive3 (ABI3) (dark-grown Arabidopsis seedlings, Rohde et    al. (2000) Plant Cell 12: 35-52), asparagine synthetase (pea root    nodules, Tsai, Coruzzi, (1990) EMBO J 9: 323-32) and mdm2 gene    (human tumor; Saucedo et al. (1998) Cell Growth Differ 9: 119-30).

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease drought or light tolerance may require up-regulation oftranscription. In contrast, transcriptional down-regulation may bedesired to inhibit those genes, transcripts and/or polypeptides thatlower such tolerance.

Typically, promoter or control elements which provide preferentialtranscription in cells, tissues or organs exposed to light producetranscript levels that are statistically significantly altered ascompared to cells, tissues or organs under decreased light exposure(intensity or length of time).

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Dark Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by dark or decreased light intensity or decreased light exposuretime can be utilized to time growth, metabolism and development and tomodulate photosynthesis capabilities for host cells or organisms. In aplant, modulation of genes, transcripts and/or polypeptides in responseto dark is useful

-   (1) to induce growth or development, such as fruit development and    maturity, despite lack of light;-   (2) to modulate genes, transcripts and/or polypeptide active at    night or on cloudy days; or-   (3) to preserve the plastid ultra structure present at the onset of    darkness.    The present promoters and control elements can also trigger response    similar to those described in the section above.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease growth and development may require up-regulation oftranscription. In contrast, transcriptional down-regulation may bedesired to inhibit those genes, transcripts and/or polypeptides thatmodulate photosynthesis capabilities.

Typically, promoter or control elements which provide preferentialtranscription under exposure to dark or decreased light intensity ordecreased exposure time produce transcript levels that are statisticallysignificantly altered.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Leaf Preferential Transcription

Promoters and control elements providing preferential transcription in aleaf can modulate growth, metabolism and development or modulate energyand nutrient utilization in host cells or organisms. In a plant,preferential modulation of genes, transcripts and/or polypeptide in aleaf is useful

-   (1) to modulate leaf size, shape, and development;-   (2) to modulate the number of leaves; or-   (3) to modulate energy or nutrient usage in relation to other organs    and tissues.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease growth may require up-regulation of transcription. In contrast,transcriptional down-regulation may be desired to inhibit energy usagein a leaf and to redirect it to the fruit instead, for instance.

Typically, promoter or control elements which provide preferentialtranscription in the cells, tissues, or organs of a leaf producetranscript levels that are statistically significantly altered ascompared to other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Root Preferential Transcription

Promoters and control elements providing preferential transcription in aroot can modulate growth, metabolism, development, nutrient uptake,nitrogen fixation or modulate energy and nutrient utilization in hostcells or organisms. In a plant, for example, preferential modulation ofgenes, transcripts, and/or in a leaf, is useful

-   (1) to modulate root size, shape, and development;-   (2) to modulate the number of roots, or root hairs;-   (3) to modulate mineral, fertilizer, or water uptake;-   (4) to modulate transport of nutrients; or-   (4) to modulate energy or nutrient usage in relation to other organs    and tissues.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease growth may require up-regulation of transcription. In contrast,transcriptional down-regulation may be desired to inhibit nutrient usagein a root and to redirect it to the leaf instead, for instance.

Typically, promoter or control elements which provide preferentialtranscription in cells, tissues or organs of a root produce transcriptlevels that are statistically significantly altered as compared to othercells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Stem/Shoot Preferential Transcription

Promoters and control elements providing preferential transcription in astem or shoot can modulate growth, metabolism and development ormodulate energy and nutrient utilization in host cells or organisms. Ina plant, preferential modulation of genes, transcripts and/or apolypeptide in a stem or shoot is useful

-   (1) to modulate stem/shoot size, shape, and development; or-   (2) to modulate energy or nutrient usage in relation to other organs    and tissues

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease growth may require up-regulation of transcription. In contrast,transcriptional down-regulation may be desired to inhibit energy usagein a stem/shoot and to redirect it to the fruit instead, for instance.

Typically, promoter or control elements which provide preferentialtranscription in the cells, tissues or organs of a stem or shoot producetranscript levels that are statistically significant as compared toother cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Fruit and Seed Preferential Transcription

Promoters and control elements providing preferential transcription in asilique or fruit can time growth, development, or maturity; or modulatefertility; or modulate energy and nutrient utilization in host cells ororganisms. In a plant preferential modulation of genes, transcriptsand/or polypeptides in a fruit is useful

-   (1) to modulate fruit size, shape, development, and maturity;-   (2) to modulate the number of fruit or seeds;-   (3) to modulate seed shattering;-   (4) to modulate components of seeds, such as, storage molecules,    starch, protein, oil, vitamins, anti-nutritional components, such as    phytic acid;-   (5) to modulate seed and/or seedling vigor or viability;-   (6) to incorporate exogenous compositions into a seed, such as    lysine rich proteins;-   (7) to permit similar fruit maturity timing for early and late    blooming flowers; or-   (8) to modulate energy or nutrient usage in relation to other organs    and tissues.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease growth may require up-regulation of transcription. In contrast,transcriptional down-regulation may be desired to inhibit late fruitmaturity, for instance.

Typically, promoter or control elements which provide preferentialtranscription in the cells, tissues or organs of siliques or fruitsproduce transcript levels that are statistically significantly alteredas compared to other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Callus Preferential Transcription

Promoters and control elements providing preferential transcription in acallus can be useful to modulating transcription in dedifferentiatedhost cells. In a plant transformation, for example, preferentialmodulation of genes or transcript in callus is useful to modulatetranscription of a marker gene, which can facilitate selection of cellsthat are transformed with exogenous polynucleotides.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease marker gene detectability may require up-regulation oftranscription. In contrast, transcriptional down-regulation may bedesired to increase the ability of the calluses to differentiate, forinstance.

Typically, promoter or control elements which provide preferentialtranscription in callus produce transcript levels that are statisticallysignificantly altered as compared to other cell types, tissues, ororgans. Calculation of P-value from the different observed transcriptlevels is one means of determining whether a promoter or control elementis providing such preferential transcription.

Usually, each P-value of the transcript levels observed in callus ascompared to at least one other cell type, tissue or organ, is less than10⁻⁴; more usually, less than 10⁻⁵; even more usually, less than 10⁻⁶;even more usually, less than 10⁻⁷ or 10⁻⁸.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Flower Specific Transcription

Promoters and control elements providing preferential transcription inflowers can modulate pigmentation or modulate fertility in host cells ororganisms. In a plant, preferential modulation of genes, transcriptsand/or polypeptides in a flower is useful,

-   (1) to modulate petal color; or-   (2) to modulate the fertility of pistil and/or stamen.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease pigmentation may require up-regulation of transcription. Incontrast, transcriptional down-regulation maybe desired to inhibitfertility, for instance.

Typically, promoter or control elements which provide preferentialtranscription in flowers produce transcript levels that arestatistically significantly altered as compared to other cells, organsor tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Immature Bud and Inflorescence Preferential Transcription

Promoters and control elements providing preferential transcription inan immature bud or inflorescence can time growth, development ormaturity or modulate fertility or viability in host cells or organisms.In a plant, preferential modulation of genes, transcripts, and/orpolypeptide in an immature bud or inflorescence is useful,

-   (1) to modulate embryo development, size, and maturity;-   (2) to modulate endosperm development, size, and composition;-   (3) to modulate the number of seeds and fruits; or-   (4) to modulate seed development and viability.

Up-regulation and down-regulation of transcription is useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease growth may require up-regulation of transcription. In contrast,transcriptional down-regulation may be desired to decrease endospermsize, for instance.

Typically, promoter or control elements which provide preferentialtranscription in immature buds and inflorescences produce transcriptlevels that are statistically significantly altered as compared to othercell types, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Senescence Preferential Transcription

Promoters and control elements providing preferential transcriptionduring senescence can be used to modulate cell degeneration, nutrientmobilization and scavenging of free radicals in host cells or organisms.Other types of responses that can be modulated include, for example,senescence associated genes (SAG) that encode enzymes thought to beinvolved in cell degeneration and nutrient mobilization (Arabidopsis;Hensel et al. (1993) Plant Cell 5: 553-64), and the CP-2/cathepsin Lgene (rat; Kim and Wright (1997) Biol Reprod 57: 1467-77). Both of thesegenes are induced during senescence.

In a plant, preferential modulation of genes, transcripts and/orpolypeptides during senescencing is useful to modulate fruit ripening.

Up-regulation and down-regulation of transcription are useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease scavenging of free radicals may require up-regulation oftranscription. In contrast, transcriptional down-regulation may bedesired to inhibit cell degeneration, for instance.

Typically, promoter or control elements which provide preferentialtranscription in cells, tissues or organs during senescence producetranscript levels that are statistically significantly altered ascompared to other conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Germination Preferential Transcription

Promoters and control elements providing preferential transcription in agerminating seed can time growth, development or maturity or modulateviability in host cells or organisms. In a plant, preferentialmodulation of genes, transcripts and/or polypeptide in a germinatingseed is useful

-   (1) to modulate the emergence of they hypocotyls, cotyledons and    radical; or-   (2) to modulate shoot and primary root growth and development;

Up-regulation and down-regulation of transcription is useful for theseapplications. For instance, genes, transcripts and/or polypeptides thatincrease growth may require up-regulation of transcription. In contrast,transcriptional down-regulation may be desired to decrease endospermsize, for instance.

Typically, promoter or control elements which provide preferentialtranscription in a germinating seed produce transcript levels that arestatistically significantly altered as compared to other cell types,organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

Results

GFP Experimental Procedures and Results Procedures

The polynucleotide sequences of the present invention were tested forpromoter activity using Green Fluorescent Protein (GFP) assays in thefollowing manner.

Approximately 1-2 kb of genomic sequence occurring immediately upstreamof the ATG translational start site of the gene of interest was isolatedusing appropriate primers tailed with BstXI restriction sites. StandardPCR reactions using these primers and genomic DNA were conducted. Theresulting product was isolated, cleaved with BstXI and cloned into theBstXI site of an appropriate vector, such as pNewBin4-HAP1-GFP (see FIG.1).

Transformation

The following procedure was used for transformation of plants

1. Stratification of WS-2 Seed.

-   Add 0.5 ml WS-2 (CS2360) seed to 50 ml of 0.2% Phytagar in a 50 ml    Corning tube and vortex until seeds and Phytagar form a homogenous    mixture.-   Cover tube with foil and stratify at 4° C. for 3 days.

2. Preparation of Seed Mixture.

-   Obtain stratified seed from cooler.-   Add seed mixture to a 1000 ml beaker.-   Add an additional 950 ml of 0.2% Phytagar and mix to homogenize.

3. Preparation of Soil Mixture.

-   Mix 24 L SunshineMix #5 soil with 16 L Therm-O-Rock vermiculite in    cement mixer to make a 60:40 soil mixture.-   Amend soil mixture by adding 2 Tbsp Marathon and 3 Tbsp Osmocote and    mix contents thoroughly.-   Add 1 Tbsp Peters fertilizer to 3 gallons of water and add to soil    mixture and mix thoroughly.-   Fill 4-inch pots with soil mixture and round the surface to create a    slight dome.-   Cover pots with 8-inch squares of nylon netting and fasten using    rubber bands.-   Place 14 4-inch pots into each no-hole utility flat.

4. Planting.

-   Using a 60 ml syringe, aspirate 35 ml of the seed mixture.-   Exude 25 drops of the seed mixture onto each pot.-   Repeat until all pots have been seeded.-   Place flats on greenhouse bench, cover flat with clear propagation    domes, place 55% shade cloth on top of flats and subirrigate by    adding 1 inch of water to bottom of each flat.

5. Plant Maintenance.

-   3 to 4 days after planting, remove clear lids and shade cloth.-   Subirrigate flats with water as needed.-   After 7-10 days, thin pots to 20 plants per pot using forceps.-   After 2 weeks, subirrigate all plants with Peters fertilizer at a    rate of 1 Tsp per gallon water.-   When bolts are about 5-10 cm long, clip them between the first node    and the base of stem to induce secondary bolts.-   6 to 7 days after clipping, perform dipping infiltration.

6. Preparation of Agrobacterium.

-   Add 150 ml fresh YEB to 250 ml centrifuge bottles and cap each with    a foam plug (Identi-Plug).-   Autoclave for 40 min at 121° C.-   After cooling to room temperature, uncap and add 0.1 ml each of    carbenicillin, spectinomycin and rifampicin stock solutions to each    culture vessel.-   Obtain Agrobacterium starter block (96-well block with Agrobacterium    cultures grown to an OD₆₀₀ of approximately 1.0) and inoculate one    culture vessel per construct by transferring 1 ml from appropriate    well in the starter block.-   Cap culture vessels and place on Lab-Line incubator shaker set at    27° C. and 250 RPM.-   Remove after Agrobacterium cultures reach an OD₆₀₀ of approximately    1.0 (about 24 hours), cap culture vessels with plastic caps, place    in Sorvall SLA 1500 rotor and centrifuge at 8000 RPM for 8 min at 4°    C.-   Pour out supernatant and put bottles on ice until ready to use.-   Add 200 ml Infiltration Media (IM) to each bottle, resuspend    Agrobacterium pellets and store on ice.

7. Dipping Infiltration.

-   Pour resuspended Agrobacterium into 16 oz polypropylene containers.-   Invert 4-inch pots and submerge the aerial portion of the plants    into the Agrobacterium suspension and let stand for 5 min.-   Pour out Agrobacterium suspension into waste bucket while keeping    polypropylene container in place and return the plants to the    upright position.-   Place 10 covered pots per flat.-   Fill each flat with 1-inch of water and cover with shade cloth.-   Keep covered for 24 hr and then remove shade cloth and polypropylene    containers.-   Resume normal plant maintenance.-   When plants have finished flowering cover each pot with a ciber    plant sleeve.-   After plants are completely dry, collect seed and place into 2.0 ml    micro tubes and store in 100-place cryogenic boxes.

Recipes:

-   0.2% Phytagar-   2 g Phytagar-   1 L nanopure water    -   Shake until Phytagar suspended    -   Autoclave 20 min

YEB (for 1 L)

-   5 g extract of meat-   5 g Bacto peptone-   1 g yeast extract-   5 g sucrose-   0.24 g magnesium sulfate    -   While stirring, add ingredients, in order, to 900 ml nanopure        water    -   When dissolved, adjust pH to 7.2    -   Fill to 1 L with nanopure water    -   Autoclave 35 min

Infiltration Medium (IM) (for 1 L)

-   2.2 g MS salts-   50 g sucrose-   5 ul BAP solution (stock is 2 mg/ml)    -   While stirring, add ingredients in order listed to 900 ml        nanopure water    -   When dissolved, adjust pH to 5.8.    -   Volume up to 1 L with nanopure water.    -   Add 0.02% Silwet L-77 just prior to resuspending Agrobacterium

High Throughput Screening—T1 Generation

1. Soil Preparation. Wear gloves at all times.

-   In a large container, mix 60% autoclaved SunshineMix #5 with 40%    vermiculite.-   Add 2.5 Tbsp of Osmocote, and 2.5 Tbsp of 1% granular Marathon per    25 L of soil.-   Mix thoroughly.

2. Fill Com-Packs With Soil.

-   Loosely fill D601 Com-Packs level to the rim with the prepared soil.-   Place filled pot into utility flat with holes, within a no-hole    utility flat.-   Repeat as necessary for planting. One flat set should contain 6    pots.

3. Saturate Soil.

-   Evenly water all pots until the soil is saturated and water is    collecting in the bottom of the flats.-   After the soil is completely saturated, dump out the excess water.

4. Plant the Seed. 5. Stratify the Seeds.

-   After sowing the seed for all the flats, place them into a dark    4° C. cooler.-   Keep the flats in the cooler for 2 nights for WS seed. Other    ecotypes may take longer. This cold treatment will help promote    uniform germination of the seed.    6. Remove Flats From Cooler and Cover With Shade Cloth. (Shade cloth    is only needed in the greenhouse)-   After the appropriate time, remove the flats from the cooler and    place onto growth racks or benches.-   Cover the entire set of flats with 55% shade cloth. The cloth is    necessary to cut down the light intensity during the delicate    germination period.-   The cloth and domes should remain on the flats until the cotyledons    have fully expanded. This usually takes about 4-5 days under    standard greenhouse conditions.

7. Remove 55% Shade Cloth and Propagation Domes.

-   After the cotyledons have fully expanded, remove both the 55% shade    cloth and propagation domes.    8. Spray Plants With Finale Mixture. Wear gloves and protective    clothing at all times.-   Prepare working Finale mixture by mixing 3 ml concentrated Finale in    48 oz of water in the Poly-TEK sprayer.-   Completely and evenly spray plants with a fine mist of the Finale    mixture.-   Repeat Finale spraying every 3-4 days until only transformants    remain. (Approximately 3 applications are necessary.)-   When satisfied that only transformants remain, discontinue Finale    spraying.

9. Weed Out Excess Transformants.

-   Weed out excess transformants such that a maximum number of five    plants per pot exist evenly spaced throughout the pot.

GFP Assay

Tissues are dissected by eye or under magnification using INOX 5 gradeforceps and placed on a slide with water and coversliped. An attempt ismade to record images of observed expression patterns at earliest andlatest stages of development of tissues listed below. Specific tissueswill be preceded with High (H), Medium (M), Low (L) designations.

Flower Pedice, I receptacle, nectary, sepal, petal, filament, anther,pollen, carpel, style, papillae, vascular, epidermis, stomata, trichomeSilique Stigma, style, carpel, septum, placentae, transmitting tissue,vascular, epidermis, stomata, abscission zone, ovule OvulePre-fertilization: inner integument,

outer integument, embryo sac,

funiculus, chalaza, micropyle, gametophyte Post-fertilization: zygote,inner integument, outer integument, seed coat, primordial, chalaza,micropyle, early endosperm, mature endosperm, embryo Embryo Suspensor,preglobular, globular, heart, torpedo, late, mature, provascular,hypophysis, radicle, cotyledons, hypocotyl Stem epidermis, cortex,vascular, xylem, phloem, pith, stomata, trichome Leaf Petiole,mesophyll, vascular, epidermis, trichome, primordial, stomata, stipule,margin

T1 Mature: These are the T1 plants resulting from independenttransformation events. These are screened between stage 6.50-6.90 (meansthe plant is flowering and that 50-90% of the flowers that the plantwill make have developed) which is 4-6 weeks of age. At this stage themature plant possesses flowers, siliques at all stages of development,and fully expanded leaves. We do not generally differentiate between6.50 and 6.90 in the report but rather just indicate 6.50. The plantsare initially imaged under UV with a Leica Confocal microscope. Thisallows examination of the plants on a global level. If expression ispresent, they are imaged using scanning laser confocal micsrocopy.

T2 Seedling: Progeny are collected from the T1 plants giving the sameexpression pattern and the progeny (T2) are sterilized and plated onagar-solidified medium containing M&S salts. In the event that there wasno expression in the T1 plants, T2 seeds are planted from all lines. Theseedlings are grown in Percival incubators under continuous light at 22°C. for 10-12 days. Cotyledons, roots, hypocotyls, petioles, leaves, andthe shoot meristem region of individual seedlings were screened untiltwo seedlings were observed to have the same pattern. Generally foundthe same expression pattern was found in the first two seedlings.However, up to 6 seedlings were screened before “no expression pattern”was recorded. All constructs are screened as T2 seedlings even if theydid not have an expression pattern in the T1 generation.

T2 Mature: The T2 mature plants were screened in a similar manner to theT1 plants. The T2 seeds were planted in the greenhouse, exposed toselection and at least one plant screened to confirm the T1 expressionpattern. In instances where there were any subtle changes in expression,multiple plants were examined and the changes noted in the tables.

T3 Seedling: This was done similar to the T2 seedlings except that onlythe plants for which we are trying to confirm the pattern are planted.

Image Data:

Images are collected by scanning laser confocal microscopy. Scannedimages are taken as 2-D optical sections or 3-D images generated bystacking the 2-D optical sections collected in series. All scannedimages are saved as TIFF files by imaging software, edited in AdobePhotoshop, and labeled in Powerpoint specifying organ and specificexpressing tissues.

Instrumentation: Microscope Inverted Leica DM IRB

-   Fluorescence filter blocks:-   Blue excitation BP 450-490; long pass emission LP 515.-   Green excitation BP 515-560; long pass emission LP 590

Objectives

-   HC PL FLUOTAR 5×/0.5-   HCPL APO 10×/0.4 IMM water/glycerol/oil-   HCPL APO 20×/0.7 IMM water/glycerol/oil-   HCXL APO 63×/1.2 IMM water/glycerol/oil

Leica TCS SP2 Confocal Scanner

-   Spectral range of detector optics 400-850 nm.-   Variable computer controlled pinhole diameter.-   Optical zoom 1-32×.-   Four simultaneous detectors:-   Three channels for collection of fluorescence or reflected light.-   One channel for transmitted light detector.-   Laser sources:-   Blue Ar 458/5 mW, 476 nm/5 mW, 488 nm/20 mW, 514 nm/20 mW.-   Green HeNe 543 nm/1.2 mW-   Red HeNe 633 nm/10 mW

Results

The section in Table 1 entitled “The spatial expression of thepromoter-marker-vector” presents the results of the GFP assays asreported by their corresponding cDNA ID number, construct number andline number. Unlike the microarray results, which measure the differencein expression of the endogenous cDNA under various conditions, the GFPdata gives the location of expression that is visible under the imagingparameters. Table 3 summarizes the results of the spatial expressionresults for each promoter.

Explanation of Table 1

Table 1 includes various information about each promoter or promotercontrol element of the invention including the nucleotide sequence, thespatial expression pattern associated with each promoter and thecorresponding results from different expression experiments.

Lengthy table referenced here US20110041208A1-20110217-T00001 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110041208A1-20110217-T00002 Pleaserefer to the end of the specification for access instructions.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110041208A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An isolated nucleic acid molecule capable of modulating transcriptionwherein the nucleic acid molecule shows at least 80% sequence identityto a sequence set forth in the Sequence Listing or a complement thereof.2. The isolated nucleic acid molecule of claim 1, wherein said nucleicacid is capable of functioning as a promoter.
 3. The isolated nucleicacid molecule of claim 1, wherein said nucleic acid molecule is capableof modulating transcription during particular developmental times or inresponse to a stimuli or in a particular cell, tissue, or organ as setforth in the Sequence Listing-Miscellaneous Feature.
 4. A vectorconstruct comprising: a) a first nucleic acid capable of modulatingtranscription wherein the nucleic acid molecule shows at least 80%sequence identity to a sequence set forth in the Sequence Listing; andb) a second nucleic acid having to be transcribed, wherein said firstand second nucleic acid molecules are heterologous to each other and areoperably linked together.
 5. A host cell comprising an isolated nucleicacid molecule according to claim 1, wherein said nucleic acid moleculeis flanked by exogenous sequence.
 6. A host cell comprising a vectorconstruct of claim
 4. 7. A method of modulating transcription bycombining, in an environment suitable for transcription: a) a firstnucleic acid molecule capable of modulating transcription wherein thenucleic acid molecule shows at least 80% sequence identity to a sequenceset forth in the Sequence Listing; and b) a second molecule to betranscribed; wherein the first and second nucleic acid molecules areheterologous to each other and operably linked together.
 8. The methodaccording to any one of claims 7, wherein said first nucleic acidmolecule is capable of modulating transcription during the particulardevelopmental times or in response to a stimuli or in a particular celltissue, or organ as set forth in the Sequence Listing-MiscellaneousFeature wherein said first nucleic acid molecule is inserted into aplant cell and said plant cell is regenerated into a plant.
 9. A plantcomprising a vector construct according to claim 4.